The paper deals with the application of Raman spectroscopy for milk fat determination. For a quality control of milk and dairy products the monitoring and analysis of milk nutritional composition is crucial. Commonly available milk was used for analyses in form of liquid milk samples and also dried milk droplets with fat concentration 0,1 %-3,5 %. Raman spectroscopy brings benefits over traditional laboratory techniques in terms of effective, rapid and reagent free way of milk fat measurements. Acquired Raman spectra were assigned to appropriate milk elements focusing on milk fat components. Method accuracy for a content of fat in milk is discussed in paper and in comparison to results obtained by conventional Röse-Gottlieb gravimetric method and butyrometry show good agreement.